Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

All animal procedures were approved by the Institutional Animal Care and Use Committee at the Los Angeles Biomedical Research Institute (protocol 011000) and carried out according to the National Institutes of Health (NIH) guidelines for the ethical treatment of animals. The mice were caged in an AAALAC-accredited facility located on the campus of Harbor-UCLA Research and Education Institute. A full-time veterinarian who specializes in laboratory animal medicine oversaw their care. Caging and husbandry was provided according to the guidelines in the US Public Health Service publication "Guide for the Care and Use of Laboratory Animals". Every attempt was made to treat the mice humanely. The survival and health of the mice was monitored three times daily. Obviously sick, lethargic mice were segregated from the group and euthanized to minimize suffering. The mice were euthanized by pentobarbital overdose (210 mg/kg), as recommended by the Panel on Euthanasia of the American Veterinary Medical Association.

1. Preparation of Tissues, Reagents and Instruments

1. Use male Balb/c mice weighting 20-22 g for all studies. Inoculate three mice per experimental group intravenously with 1 x 10⁶ yeast-phase C. albicans cells. Sacrifice animals and harvest kidneys at specific time points post-infection. Place each kidney into a screw cap tube, snap freeze in liquid nitrogen, and store at -80 °C for later RNA extraction⁶.
   Note: The animal experiments were carried out as described in detail in Xu et al.⁶.
2. Prepare the following reagents and instruments before removing kidneys from the -80 °C freezer:
   1. Open a total RNA extraction kit. Add 2-mercaptoethanol (1% v/v) to buffer RLT and mix well (prepare 2 ml for each sample).
   2. Prepare phenol : chloroform : isoamyl alcohol 25:24:1 (need 600 µl for each sample).
   3. Label M-type homogenization tubes (M-tube) and chill on ice.
   4. Label 2 ml screw cap tubes, and add approximately 300 µl Zirconia beads to each tube.
   5. Check instruments are available: tissue dissociator, beadbeater, tabletop centrifuge with adaptors for 50 ml tubes and 96 well plates, photometer.

2. RNA Extraction from Infected Tissues 

1. Remove tubes containing kidneys from -80 °C freezer and put on ice. Add 1.2 ml buffer RLT with 2-mercaptoethanol to each kidney. Decant kidney with buffer into an M-type homogenization tubes.
2. Homogenize the kidney using a tissue dissociator on pre-loaded setting RNA_02.01.
3. Centrifuge at 1,000 x g for 1 min in a tabletop centrifuge at RT.
4. Transfer 600 µl homogenate to the screw cap tube containing Zirconia beads. Save the remaining homogenate on ice.
5. Add 600 µl phenol : chloroform : isoamyl alcohol 25:24:1 to the tubes from the previous step.
6. Close the lids tightly and vortex on beadbeater for 3 min in a 4 °C cold room.
7. Centrifuge the tubes at 15,000 x g for 5 min in a 4 °C cold room.
8. Carefully transfer the aqueous phase to a new 1.5 ml microfuge tube, mix well with equal volume of 70% ethanol, then load onto the spin column (load twice).
9. Wash the spin column once with 700 µl buffer RW, followed by twice with 500 µl buffer RPE. Centrifuge one extra minute in a dry collection tube to remove remaining liquid in the spin column. Perform all centrifugation steps at 8,000 x g.
10. Elute RNA with 50 µl H₂O. Measure RNA concentration at OD₂₆₀ using a spectrophotometer.

3. Gene Expression Profiling Using Digital Bar-coding

1. Thaw reporter codeset (green cap tube) and capture codeset (gray cap tube) on ice.
2. Add 130 µl hybridization buffer to the reporter codeset, invert to mix and spin down.
3. Add 20 µl of the mix to each of the 12 reaction tubes.
4. Add 10 µg of total tissue RNA (in a volume of 5 µl) to each tube. Mix by pipetting.
5. Add 5 µl capture codeset to each tube. Mix by flipping the tube followed by a quick spin down.
6. Incubate the reactions at 65 °C in a thermal cycler with heated lid O/N (~18 hr).
7. Remove one sample cartridge from -20 °C and let it warm to RT.
8. Remove two reagent plates from 4 °C and centrifuge at 670 x g for 2 min in a tabletop centrifuge.
9. Set up the prep station following step-by-step instructions on the screen, select the high sensitivity option.
10. Remove the reactions from the thermal cycler and immediately load on the prep station. Select to run the high sensitivity program (3 hr).
11. When the prep station program is complete, remove the cartridge and seal the lanes with a clear tape. Apply mineral oil to the bottom of the cartridge (for generation one nCounter only, later generations do not require this step), then load the cartridge onto the digital analyzer.
12. Set up the digital analyzer following step-by-step instructions on the screen, select the high resolution scan option.
13. Select the high resolution (600 fields) option, run the scanning program (~4.5 hr).
14. When the scanning program is complete, download the results (or choose to receive the results by email), and import raw data into manufacturer's software. The software will automatically check data quality and raise flags if the quality of the data falls out of the normal range. Perform technical adjustment using the built-in function (optional, follow instructions in the software), then export the data as an excel file.
15. Normalize the data using one of the following methods: total counts from all genes in the codeset; one or a few internal control genes; geometric mean of highly expressed genes (see discussion).
16. Calculate the mean expression values for each gene (if the experiment was done in replicates or triplicates), then calculate the ratio of expression levels among different experimental groups.
17. (optional) Analyze and visualize the profiling results by built-in functions in the nSolver software or a clustering program such as MultiExperiment Viewer¹⁰.